High-efficiency three-dimensional cultivation method of antler-like mushroom based on multi-stage temperature and humidity control

Abstract

This invention relates to the field of edible mushroom cultivation technology, specifically to a high-efficiency three-dimensional cultivation method for *Pleurotus ostreatus* based on multi-stage temperature and humidity control, comprising the following steps: S1, substrate pretreatment; S2, mycelial culture; S3, mycelial physiological activity detection; S4, dynamic regulation of environmental parameters; S5, enzyme induction treatment during the color change stage; S6, directional bud induction treatment; S7, gradient development regulation; S8, pre-harvest regulation. This invention, by establishing a three-dimensional cultivation system combining multi-stage dynamic temperature and humidity control with mycelial physiological state monitoring, achieves precise environmental adaptation at each stage of mycelial culture, color change induction, and fruiting body development. It effectively solves the technical problems of mismatched environmental parameters and growth requirements, and imbalanced interlayer microenvironments in traditional cultivation, significantly improving the cultivation efficiency and quality consistency of *Pleurotus ostreatus*.

Description

Technical Field

[0001] This invention relates to the field of edible fungi cultivation technology, and in particular to a high-efficiency three-dimensional cultivation method for *Pleurotus ostreatus* based on multi-stage temperature and humidity control. Background Technology

[0002] In the industrialized cultivation of *Pleurotus ostreatus*, there is a significant contradiction between the environmental requirements for mycelial growth and fruiting body development. Traditional cultivation methods, which employ constant temperature and humidity, struggle to meet the differentiated needs of different growth stages, leading to uneven mycelial maturity, prolonged color change period, and asynchronous fruiting body development. This is particularly pronounced under three-dimensional cultivation conditions, where uneven temperature and humidity distribution between layers and CO2 accumulation severely impact mushroom quality and yield. While existing technologies attempt to improve cultivation through environmental control, significant shortcomings remain in areas such as determining mycelial physiological state and coupling control of multi-stage environmental parameters, resulting in low cultivation efficiency.

[0003] To address the above problems, existing technologies mainly suffer from the following deficiencies: (1) a lack of precise criteria for determining mycelial maturity, making it difficult to adjust cultivation parameters in a timely manner; (2) imprecise environmental control between layers of the three-dimensional cultivation rack, resulting in uneven fruiting body development due to temperature and humidity gradient imbalance; and (3) a lack of scientific methods for controlling moisture content before harvesting, affecting the marketability of the mushrooms. Therefore, it is urgent to develop a three-dimensional cultivation method based on multi-stage temperature and humidity control to solve the above problems. Summary of the Invention

[0004] To achieve the above objectives, this invention provides a highly efficient three-dimensional cultivation method for *Pleurotus ostreatus* based on multi-stage temperature and humidity control.

[0005] A high-efficiency three-dimensional cultivation method for *Pleurotus ostreatus* based on multi-stage temperature and humidity control includes the following steps:

[0006] S1, Substrate pretreatment: The cultivation substrate is crushed and mixed with sawdust, wheat bran, soybean meal and calcium carbonate, and then steam sterilized to obtain the pretreated substrate;

[0007] S2, Mycelial Culture Stage: The pretreated substrate after inoculation is placed in a three-dimensional cultivation rack culture chamber, and dynamic temperature drop culture is carried out. At the same time, intermittent blue light irradiation is performed, and laccase activity and extracellular polysaccharide content are detected by a metabolite sensor to generate mycelial maturity index.

[0008] S3, Mycelial Physiological Activity Detection: When the mycelial maturity index reaches the set threshold, the mycelial ATP content is measured. Cultivation bags that meet the requirements enter the next stage, while those that do not meet the requirements are returned for continued cultivation.

[0009] S4, Dynamic control of environmental parameters: The three-dimensional cultivation rack is divided into different temperature and humidity control zones according to the mycelial maturity index, and an interlayer environmental gradient is constructed by controlling the temperature difference between layers and adjusting the flow plate.

[0010] S5, Enzyme induction treatment during the color change period: Spray a compound enzyme inducer on the surface of the cultivation bag in the mature area, and at the same time start the longitudinal airflow circulation to maintain the CO2 concentration within the target range;

[0011] S6, Targeted bud induction treatment: Open oxygen vents on the surface of the cultivation bag after color change, and use intermittent atomization to maintain the surface moisture of the mycelium;

[0012] S7, Gradient Development Regulation: Establish temperature and humidity gradients in the vertical direction of the three-dimensional cultivation rack to promote uniform development of fruiting bodies;

[0013] S8, Pre-harvest conditioning: When the fruiting bodies reach the harvesting standard, low-temperature airflow drying is used to regulate the moisture content of the stipe before harvesting.

[0014] Optionally, S1 specifically includes:

[0015] S11, Raw material crushing and processing: The sawdust is crushed to a particle size of 2-3mm using a twin-shaft crusher, and the bran and soybean meal are passed through a 40-mesh sieve respectively;

[0016] S12, Matrix formulation and mixing: Mix 65% sawdust, 20% wheat bran, 12% soybean meal and 3% calcium carbonate by weight percentage to form a mixed matrix;

[0017] S13, Pulse Steam Sterilization: The mixed substrate is loaded into the culture chamber, saturated steam is introduced to raise the pressure to 0.15-0.18 MPa, and maintained for 35-45 minutes. After sterilization, the pressure is naturally reduced to normal pressure.

[0018] S14, Moisture content control: Spray sterile water onto the sterilized substrate to adjust the moisture content to 62-65% to obtain the pretreated substrate.

[0019] Optionally, S2 specifically includes:

[0020] S21, Loading the culture chamber: Spread the pretreated substrate after inoculation evenly in the culture chamber of the three-dimensional cultivation rack, and control the thickness of the substrate layer to 8-10cm.

[0021] S22, dynamic temperature drop culture: maintain the culture chamber temperature at 24-26℃ for the first 5 days, and then decrease the temperature by 0.7-0.9℃ daily from the 6th day until the temperature drops to 18-19℃, while maintaining the air humidity at 75-78% throughout the process;

[0022] S23, Intermittent Blue Light Control: Turn on the top-mounted LED light source for blue light irradiation daily, with a wavelength of 450-460nm and a light intensity of 750-850lux. The total irradiation time is 10-12 hours per day, and the light source is turned off for 0.5 hours after every 2 hours of irradiation.

[0023] S24, Metabolite detection: Laccase activity is detected every 24 hours using a laccase activity sensor at the bottom of the culture chamber, while the extracellular polysaccharide content is detected simultaneously using a microfluidic chip. When the laccase activity is ≥35U / g and the extracellular polysaccharide is ≥18mg / g, a signal indicating that the mycelial maturity has been achieved is generated.

[0024] Optionally, S3 specifically includes:

[0025] S31, Mycelial Sample Collection: After the mycelial maturity standard signal is triggered, take a 0.5-1.0g sample from the middle of the mycelial layer of the cultivation bag, at a sampling depth of 1 / 3 of the substrate thickness.

[0026] S32, Sample pretreatment: Mix the sample with sterile physiological saline at a mass ratio of 1:4, centrifuge at 8000-10000 rpm for 10-15 minutes at 4-6℃, and take the supernatant.

[0027] S33, ATP content detection: Add ATP lysis buffer to the supernatant, with a volume ratio of lysis buffer to supernatant of 1:2. After shaking and mixing, let stand for 5-8 minutes, then inject into a bio-fluorescence detector to measure the fluorescence value.

[0028] S34, Threshold determination: When the detected ATP content is ≥1.0×10⁻ 8 When the concentration is less than mol / g, it is marked as a qualified cultivation package; the detected value is <1.0×10⁻ 8 Individuals with a concentration of mol / g are marked as not meeting the standard;

[0029] S35, Re-cultivation treatment: The substandard cultivation package was returned to the S2 stage for 3 more days of cultivation, during which 0.5 hours of blue light irradiation was added daily, and the cultivation temperature was reduced by 0.3℃ compared to the original stage.

[0030] Optionally, S4 specifically includes:

[0031] S41, Cultivation rack zoning: The vertical cultivation rack is divided into three control zones: the upper zone is the active zone, with a height of 1.2-1.5m; the middle zone is the transition zone, with a height of 0.8-1.2m; and the lower zone is the maturity zone, with a height of 0.5-0.8m.

[0032] S42, temperature and humidity parameter settings: active zone set temperature 20℃, relative humidity 80%, transition zone set temperature 19℃, relative humidity 75%, mature zone set temperature 18℃, relative humidity 70%;

[0033] S43, Baffle Angle Adjustment: The angle of the interlayer baffle is automatically adjusted according to real-time CO2 concentration data. When the CO2 concentration is >500ppm, the baffle opening angle increases by 5-10°; when the CO2 concentration is ≤450ppm, the baffle opening angle decreases by 5-10°.

[0034] S44, Environmental parameter monitoring: Collect temperature, humidity and CO2 concentration data for each control zone every 30 minutes. Temperature measurement accuracy is ±0.1℃, humidity measurement accuracy is ±1%, and CO2 concentration measurement range is 0-2000ppm.

[0035] Optionally, S5 specifically includes:

[0036] S51, Preparation of inducer: Mix lignin peroxidase and laccase at a ratio of 1:1.5 enzyme activity units, add 0.05% Tween-80 as a surfactant, and prepare a compound enzyme inducer solution with a total enzyme activity of 0.15-0.25 U / mL;

[0037] S52, Spraying treatment: Use a rotating atomizing nozzle to evenly spray the surface of the mature cultivation bag, with a spraying volume of 15-20 mL / m², a nozzle rotation speed of 3000-3500 rpm, and an atomized particle size of 50-80 μm.

[0038] S53, Airflow circulation start: Turn on the axial flow fans at the top and bottom of the three-dimensional cultivation rack to form a longitudinal airflow of 0.5-0.8m / s, with the airflow circulating from top to bottom;

[0039] S54, CO2 concentration control: The concentration is monitored in real time by infrared CO2 sensors installed on each layer of the cultivation rack. When the detected value is >500ppm, the fan speed is increased by 10-15%, and when the detected value is <450ppm, the fan speed is decreased by 5-10%.

[0040] S55, Environmental balance maintenance: Keep the environment still for 30-45 minutes after induction treatment, during which time the temperature is maintained at 18-20℃ and the relative humidity at 68-72%.

[0041] Optionally, S6 specifically includes:

[0042] S61, Aeration port opening: Use an 8-10mm diameter ring punch to make cross-shaped aeration ports on the surface of the cultivation bag, with an opening depth of 1.5-2.0cm. Make 4-6 aeration ports on each cultivation bag, and keep the distance between adjacent aeration ports 5-8cm.

[0043] S62, Atomization Preparation: Configure an ultrasonic atomization device, set the atomization frequency to 28-32kHz, and control the atomized particle diameter to 10-15μm;

[0044] S63, intermittent atomization control: adopts a pulse mode with a working cycle of 8-10 seconds of spraying and 15-20 seconds of intermittent spraying, and the atomization volume is controlled at 0.5-0.8 mL / time·m²;

[0045] S64, Moisture Monitoring: Real-time monitoring of mycelial surface moisture content using an infrared moisture sensor, maintaining surface moisture content at 65-68%;

[0046] S65, Environmental Coordination: During atomization, maintain the cultivation environment temperature at 20-22℃, the relative humidity at 80-85%, and the CO2 concentration at 400-450ppm.

[0047] Optionally, S7 specifically includes:

[0048] S71, Gradient Parameter Settings: Set three temperature and humidity gradients in the vertical direction of the three-dimensional cultivation rack. The top temperature is set to 22℃, the middle temperature to 20℃, and the bottom temperature to 18℃; the top humidity is set to 88%, the middle humidity to 82%, and the bottom humidity to 78%.

[0049] S72, Airflow organization and control: Adjustable ventilation holes are set on the side of the cultivation rack, with the upper ventilation holes opening at 30-40%, the middle at 20-30%, and the lower at 10-20%, forming natural convection from top to bottom.

[0050] Optionally, S8 specifically includes:

[0051] S81, Harvesting criteria: When the diameter of the fruiting body cap reaches 3.2-3.8cm and the edge of the cap changes from inward rolling to flat, it is judged to meet the harvesting criteria;

[0052] S82, Preparation of airflow drying system: Start the refrigeration unit to pre-cool the airflow temperature to 4-6℃ lower than the ambient temperature, adjust the airflow speed to 0.8-1.2m / s, and convey the airflow downward at a 45° angle to the cultivation rack;

[0053] S83, gradient drying process: drying is carried out in two stages: the first stage lasts for 1-6 hours, maintaining the airflow temperature at 16-18℃; the second stage lasts for 6-12 hours, reducing the temperature to 14-16℃.

[0054] S84, Moisture content control: The moisture content of the stipe is monitored in real time by a near-infrared moisture meter, and drying is terminated when the detected value drops to 68-70%.

[0055] S85, Harvesting operation: Use a rotary cutter to harvest along the surface of the cultivation bag at a depth of 0.5-1.0cm. After harvesting, immediately transfer to a pre-cooling room at 4-6℃ for temporary storage.

[0056] The beneficial effects of this invention are:

[0057] This invention effectively solves the problem of mismatch between environmental parameters and growth requirements in traditional cultivation by combining multi-stage dynamic temperature and humidity control with mycelial physiological state monitoring. By precisely regulating the environmental conditions of key stages such as mycelial culture, color change induction, and fruiting body development, it significantly improves the uniformity of mycelial growth and the synchronicity of fruiting body development, avoiding growth obstacles caused by environmental fluctuations.

[0058] This invention, through the establishment of a three-dimensional cultivation environment gradient control system, fundamentally overcomes the technical problem of interlayer microenvironment imbalance; by coordinating the coupling effects of temperature, humidity, airflow and CO2 concentration, it not only optimizes the environmental adaptability of each growth stage, but also achieves precise control of harvest quality, providing reliable technical support for the industrialized cultivation of deer antler mushrooms. Attached Figure Description

[0059] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only for this invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0060] Figure 1 This is a schematic diagram of the efficient three-dimensional cultivation method for *Agaricus esculentus* according to an embodiment of the present invention;

[0061] Figure 2 This is a schematic diagram of the dynamic control method for environmental parameters according to an embodiment of the present invention. Detailed Implementation

[0062] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. It should also be noted that, to make the embodiments more comprehensive, the following embodiments are the best and preferred embodiments, and those skilled in the art can use other alternative methods to implement some well-known technologies; moreover, the accompanying drawings are only for more specific description of the embodiments and are not intended to specifically limit the present invention.

[0063] It should be noted that the use of terms such as "an embodiment," "an embodiment," "an exemplary embodiment," and "some embodiments" in the specification indicates that the described embodiment may include a specific feature, structure, or characteristic, but not every embodiment necessarily includes that specific feature, structure, or characteristic. Furthermore, when a specific feature, structure, or characteristic is described in connection with an embodiment, implementing such a feature, structure, or characteristic in conjunction with other embodiments (whether explicitly described or not) should be within the knowledge of those skilled in the art.

[0064] Generally, terms can be understood at least partly from their use in context. For example, depending at least partly on the context, the term "one or more" as used herein can be used to describe any feature, structure, or characteristic in a singular sense, or a combination of features, structures, or characteristics in a plural sense. Additionally, the term "based on" can be understood not necessarily to convey an exclusive set of factors, but rather, alternatively, depending at least partly on the context, to allow for the presence of other factors that are not necessarily explicitly described. Example 1

[0065] like Figures 1-2 As shown, the efficient three-dimensional cultivation method of *Pleurotus ostreatus* based on multi-stage temperature and humidity control includes the following steps:

[0066] S1, Substrate pretreatment: The cultivation substrate is crushed and mixed with sawdust, wheat bran, soybean meal and calcium carbonate, and then steam sterilized to obtain the pretreated substrate;

[0067] S2, Mycelial Culture Stage: The pretreated substrate after inoculation is placed in a three-dimensional cultivation rack culture chamber, and dynamic temperature drop culture is carried out. At the same time, intermittent blue light irradiation is performed, and laccase activity and extracellular polysaccharide content are detected by a metabolite sensor to generate mycelial maturity index.

[0068] S3, Mycelial Physiological Activity Detection: When the mycelial maturity index reaches the set threshold, the mycelial ATP content is measured. Cultivation bags that meet the requirements enter the next stage, while those that do not meet the requirements are returned for continued cultivation.

[0069] S4, Dynamic control of environmental parameters: The three-dimensional cultivation rack is divided into different temperature and humidity control zones according to the mycelial maturity index, and an interlayer environmental gradient is constructed by controlling the temperature difference between layers and adjusting the flow plate.

[0070] S5, Enzyme induction treatment during the color change period: Spray a compound enzyme inducer on the surface of the cultivation bag in the mature area, and at the same time start the longitudinal airflow circulation to maintain the CO2 concentration within the target range;

[0071] S6, Targeted bud induction treatment: Open oxygen vents on the surface of the cultivation bag after color change, and use intermittent atomization to maintain the surface moisture of the mycelium;

[0072] S7, Gradient Development Regulation: Establish temperature and humidity gradients in the vertical direction of the three-dimensional cultivation rack to promote uniform development of fruiting bodies;

[0073] S8, Pre-harvest conditioning: When the fruiting bodies reach the harvesting standard, low-temperature airflow drying is used to regulate the moisture content of the stipe before harvesting.

[0074] S1 specifically includes:

[0075] S11, Raw material crushing and processing: The sawdust is crushed to a particle size of 2.5mm using a twin-shaft crusher, and the bran and soybean meal are passed through a 40-mesh sieve respectively;

[0076] S12, Matrix formulation and mixing: Mix 65% sawdust, 20% wheat bran, 12% soybean meal and 3% calcium carbonate by weight percentage to form a mixed matrix;

[0077] S13, Pulse Steam Sterilization: The mixed substrate is loaded into the culture chamber, saturated steam is introduced to raise the pressure to 0.17 MPa, and maintained for 40 minutes. After sterilization, the pressure is naturally reduced to normal pressure.

[0078] S14, Moisture content control: Spray sterile water onto the sterilized substrate to adjust the moisture content to 63% to obtain the pretreated substrate.

[0079] S2 specifically includes:

[0080] S21, Loading the culture chamber: Spread the pretreated substrate after inoculation evenly in the culture chamber of the three-dimensional cultivation rack, and control the thickness of the substrate layer to 9cm.

[0081] S22, dynamic temperature drop culture: maintain the culture chamber temperature at 25℃ for the first 5 days, and then decrease the temperature by 0.8℃ daily from the 6th day until the temperature drops to 18.5℃, while maintaining an air humidity of 77% throughout the process;

[0082] S23, Intermittent Blue Light Control: The top-mounted LED light source is turned on daily for blue light irradiation, with a wavelength of 455nm and a light intensity of 800lux. The total irradiation time is 11 hours per day, and the light source is turned off for 0.5 hours after every 2 hours of irradiation.

[0083] S24, Metabolite detection: Laccase activity is detected every 24 hours using a laccase activity sensor at the bottom of the culture chamber, while the extracellular polysaccharide content is detected simultaneously using a microfluidic chip. When the laccase activity is ≥35U / g and the extracellular polysaccharide is ≥18mg / g, a signal indicating that the mycelial maturity has been achieved is generated.

[0084] S3 specifically includes:

[0085] S31, Mycelial Sample Collection: After the mycelial maturity standard signal is triggered, take 0.8g of sample from the middle of the mycelial layer of the cultivation bag, at a sampling depth of 1 / 3 of the material layer thickness.

[0086] S32, Sample pretreatment: Mix the sample with sterile physiological saline at a mass ratio of 1:4, centrifuge at 9000 rpm for 12 minutes at 5℃, and take the supernatant.

[0087] S33, ATP content detection: Add ATP lysis buffer to the supernatant, with a volume ratio of lysis buffer to supernatant of 1:2. After shaking and mixing, let stand for 6 minutes and inject into a bio-fluorescence detector to measure the fluorescence value.

[0088] S34 has an ATP content of 1.5 × 10⁻ 8 If the concentration is mol / g, it is marked as a qualified cultivation package.

[0089] S4 specifically includes:

[0090] S41, Cultivation rack zoning: The vertical cultivation rack is divided into three control zones: the upper zone is the active zone with a height of 1.3m; the middle zone is the transition zone with a height of 1.0m; and the lower zone is the maturity zone with a height of 0.6m.

[0091] S42, temperature and humidity parameter settings: active zone set temperature 20℃, relative humidity 80%, transition zone set temperature 19℃, relative humidity 75%, mature zone set temperature 18℃, relative humidity 70%;

[0092] S43, Baffle Angle Adjustment: The angle of the interlayer baffle is automatically adjusted according to real-time CO2 concentration data. When the CO2 concentration is >500ppm, the baffle opening angle increases by 5-10°; when the CO2 concentration is ≤450ppm, the baffle opening angle decreases by 5-10°.

[0093] S44, Environmental parameter monitoring: Collect temperature, humidity and CO2 concentration data for each control zone every 30 minutes. Temperature measurement accuracy is ±0.1℃, humidity measurement accuracy is ±1%, and CO2 concentration measurement range is 0-2000ppm.

[0094] S5 specifically includes:

[0095] S51, Preparation of inducer: Mix lignin peroxidase and laccase at a ratio of 1:1.5 enzyme activity units, add 0.05% Tween-80 as a surfactant, and prepare a compound enzyme inducer solution with a total enzyme activity of 0.2 U / mL.

[0096] S52, Spraying treatment: Use a rotating atomizing nozzle to spray the surface of the mature cultivation bag evenly, with a spraying volume of 18mL / m², a nozzle speed of 3300rpm, and an atomized particle size of 60μm.

[0097] S53, Airflow circulation start: Turn on the axial flow fans at the top and bottom of the three-dimensional cultivation rack to form a longitudinal airflow of 0.6m / s, with the airflow circulating from top to bottom;

[0098] S54, CO2 concentration control: The concentration is monitored in real time by infrared CO2 sensors installed on each layer of the cultivation rack. When the detected value is >500ppm, the fan speed is increased by 12%; when the detected value is <450ppm, the fan speed is decreased by 8%.

[0099] S55, Environmental balance maintenance: Keep the environment still for 40 minutes after induction treatment, during which time the temperature is maintained at 19℃ and the relative humidity at 70%.

[0100] S6 specifically includes:

[0101] S61, Aeration port opening: Use a 9mm diameter ring punch to make cross-shaped aeration ports on the surface of the cultivation bag, with an opening depth of 1.8cm. Make 5 aeration ports on each cultivation bag, and keep the distance between adjacent aeration ports 6cm.

[0102] S62, Atomization Preparation: Configure an ultrasonic atomization device, set the atomization frequency to 30kHz, and control the atomized particle diameter to 12μm;

[0103] S63, intermittent atomization control: adopts a pulse mode with a working cycle of 9 seconds of spraying and 18 seconds of intermittent spraying, and the atomization volume is controlled at 0.7 mL / time·m²;

[0104] S64, Moisture Monitoring: Real-time monitoring of mycelial surface moisture content using an infrared moisture sensor, maintaining surface moisture content at 66%;

[0105] S65, Environmental Coordination: During atomization, maintain the cultivation environment temperature at 21℃, relative humidity at 83%, and CO2 concentration at 420ppm.

[0106] S7 specifically includes:

[0107] S71, Gradient Parameter Settings: Set three temperature and humidity gradients in the vertical direction of the three-dimensional cultivation rack. The top temperature is set to 22℃, the middle temperature to 20℃, and the bottom temperature to 18℃; the top humidity is set to 88%, the middle humidity to 82%, and the bottom humidity to 78%.

[0108] S72, Airflow organization and control: Adjustable ventilation holes are set on the side of the cultivation rack, with the upper ventilation hole opening at 35%, the middle at 25%, and the lower at 15%, forming natural convection from top to bottom.

[0109] S8 specifically includes:

[0110] S81, Harvesting criteria: When the diameter of the fruiting body cap reaches 3.5cm and the edge of the cap changes from inward to flat, it is judged to meet the harvesting criteria;

[0111] S82, Preparation of airflow drying system: Start the refrigeration unit to pre-cool the airflow temperature to 5°C lower than the ambient temperature, adjust the airflow speed to 0.9m / s, and convey the airflow downward at a 45° angle to the cultivation rack;

[0112] S83, gradient drying process: drying is carried out in two stages: the first stage lasts for 3 hours, maintaining the airflow temperature at 17°C; the second stage lasts for 8 hours, reducing the temperature to 15°C.

[0113] S84, Moisture content control: The moisture content of the stipe is monitored in real time by a near-infrared moisture meter, and drying is terminated when the detected value drops to 69%.

[0114] S85, Harvesting operation: Use a rotary cutter to harvest along the surface of the cultivation bag at 0.8cm. After harvesting, immediately transfer to a 5℃ pre-cooling room for temporary storage. Example 2

[0115] S1: The sawdust is crushed to a particle size of 2mm using a twin-shaft pulverizer. The bran and soybean meal are passed through a 40-mesh sieve and then mixed with calcium carbonate to form a mixed matrix. The mixed matrix is ​​then placed into a sterilization chamber for pulse steam sterilization. The steam pressure is controlled at 0.15MPa and the sterilization time is 35 minutes. After sterilization, the pressure is naturally reduced to atmospheric pressure, and sterile water is sprayed into it to adjust the moisture content to 62%, thus obtaining the pretreated matrix.

[0116] S2: The pretreated substrate after inoculation is evenly spread in the culture chamber of the three-dimensional cultivation rack, and the thickness of the substrate layer is controlled at 8cm. The culture temperature is maintained at 24℃ for the first 5 days. From the 6th day, the temperature is reduced by a gradient of 0.7℃ every day until the temperature drops to 18℃. The relative humidity is maintained at 75% throughout the process. Simultaneously, a blue light source with a wavelength of 450nm and a light intensity of 750lux is set up for intermittent irradiation, with an irradiation time of 10 hours per day.

[0117] S3: After the mycelial maturity threshold signal is triggered, take a 0.5g sample from the middle of the mycelial layer on the cultivation bag; mix the sample with sterile physiological saline at a mass ratio of 1:4, centrifuge at 8000rpm for 10 minutes at 4℃, collect the supernatant and add lysis buffer at a volume ratio of 1:2, vortex to mix, let stand for 5 minutes, and then inject into a biofluorescence detector to measure the ATP fluorescence value; the ATP content is 1.8×10⁻ 8 mol / g;

[0118] S4: The vertical cultivation rack is divided into three control zones: an upper active zone (1.2m high), a middle transition zone (0.8m high), and a lower maturation zone (0.5m high). The environmental parameters for each zone are set as follows: active zone temperature 20℃, humidity 80%; transition zone temperature 19℃, humidity 75%; maturation zone temperature 18℃, humidity 70%. CO2 concentration data for each layer is collected by a CO2 infrared sensor, and the angle of the guide plate is controlled in real time.

[0119] S5: The prepared compound enzyme inducer was made by mixing lignin peroxidase and laccase at an enzyme activity ratio of 1:1.5, and adding 0.05% Tween-80 to adjust the total enzyme activity to 0.15 U / mL. A rotating atomizer at 3000 rpm was used to evenly spray the surface of the mature cultivation bags, with a spray volume of 15 mL / m² and atomized particle size controlled at 50 μm. Then, the axial flow fans at the top and bottom of the three-dimensional cultivation rack were activated to create a longitudinal airflow of 0.5 m / s, circulating from top to bottom. When the CO2 concentration was higher than 500 ppm, the fan speed was increased by 10%, and when it was lower than 450 ppm, the fan speed was reduced by 5%. After spraying, the mixture was left to stand for 35 minutes, maintaining a temperature of 18℃ and a humidity of 68%.

[0120] S6: Four cross-shaped aeration ports were made on the surface of each cultivation bag using an 8mm diameter annular punch, with an opening depth of 1.5cm and a spacing of 5cm between adjacent ports; an ultrasonic atomizing device with a 28kHz atomization frequency and a 10μm atomized particle diameter was configured; an intermittent pulse mode of 8 seconds of spraying and 15 seconds of rest was adopted, with the atomization volume controlled at 0.5mL / time·m²; the surface moisture content of the mycelium was monitored in real time using an infrared moisture sensor and stabilized at 65%; the ambient temperature was maintained at 20℃, the relative humidity at 80%, and the CO2 concentration at 400ppm during the process.

[0121] S7: Construct a three-layer temperature and humidity gradient in the vertical direction of the cultivation rack: top layer temperature 22℃, middle layer temperature 20℃, and bottom layer temperature 18℃; top layer humidity set at 88%, middle layer humidity at 82%, and bottom layer humidity at 78%; adjustable ventilation holes are installed on the side walls of the cultivation rack, with ventilation openings according to the layers: 30% for the upper layer, 20% for the middle layer, and 10% for the lower layer. This airflow organization forms a natural convection microenvironment from top to bottom to guide the uniform development of fruiting bodies.

[0122] S8: When the fruiting body cap diameter reaches 3.2cm and the edge changes from inward rolling to flat, it is considered to have met the harvesting standard; start the refrigeration unit to pre-cool the airflow to 4℃ lower than the cultivation environment, adjust the wind speed to 0.8m / s, and blow the airflow downward at a 45° angle onto the cultivation rack; the drying process is carried out in two stages, the first stage lasts for 1 hour, the airflow temperature is maintained at 16℃, and the second stage lasts for 6 hours, the air temperature is adjusted to 14℃; monitor the moisture content of the stipe in real time using a near-infrared moisture detector, and stop the drying process when the moisture content drops to 68%; finally, use a rotating cutting knife to harvest along 0.5cm from the surface of the cultivation bag, and immediately transfer the harvested fruiting bodies to a 4℃ pre-cooling temporary storage room for preservation. Example 3

[0123] S1: The sawdust is crushed to a particle size of 3mm using a twin-shaft pulverizer. The bran and soybean meal are passed through a 40-mesh sieve and then mixed with calcium carbonate to form a mixed matrix. The mixed matrix is ​​then placed into a sterilization chamber for pulsed steam sterilization. The steam pressure is controlled at 0.18MPa and the sterilization time is 45 minutes. After sterilization, the pressure is naturally reduced to atmospheric pressure, and sterile water is sprayed into it to adjust the moisture content to 65%, thus obtaining the pretreated matrix.

[0124] S2: The pretreated substrate after inoculation is evenly spread in the culture chamber of the three-dimensional cultivation rack, and the thickness of the substrate layer is controlled at 10cm. The culture temperature is maintained at 26℃ for the first 5 days. From the 6th day, the temperature is reduced by a gradient of 0.9℃ every day until the temperature drops to 19℃. The relative humidity is maintained at 78% throughout the process. Simultaneously, a blue light source with a wavelength of 460nm and a light intensity of 850lux is set up for intermittent irradiation, with an irradiation time of 12 hours per day.

[0125] S3: After the mycelial maturity threshold signal is triggered, take 1.0g of sample from the middle of the mycelial layer on the cultivation bag; mix the sample with sterile physiological saline at a mass ratio of 1:4, centrifuge at 10000rpm for 15 minutes at 6℃, collect the supernatant and add lysis buffer at a volume ratio of 1:2, vortex to supernatant, let stand for 8 minutes, and then inject into a biofluorescence detector to measure the ATP fluorescence value; the ATP content is 1.3×10⁻ 8 mol / g;

[0126] S4: The vertical cultivation rack is divided into three control zones: an upper active zone (1.5m high), a middle transition zone (1.2m high), and a lower maturation zone (0.8m high). The environmental parameters for each zone are set as follows: active zone temperature 20℃, humidity 80%; transition zone temperature 19℃, humidity 75%; maturation zone temperature 18℃, humidity 70%. CO2 concentration data for each layer is collected by a CO2 infrared sensor, and the angle of the guide plate is controlled in real time.

[0127] S5: The prepared compound enzyme inducer was a mixture of lignin peroxidase and laccase at an enzyme activity ratio of 1:1.5, with the addition of 0.05% Tween-80 to adjust the total enzyme activity to 0.25 U / mL. A rotating atomizer at 3500 rpm was used to evenly spray the surface of the mature cultivation bags, with a spray volume of 20 mL / m² and atomized particle size controlled at 80 μm. Subsequently, the axial flow fans at the top and bottom of the three-dimensional cultivation rack were activated to create a longitudinal airflow of 0.8 m / s, circulating from top to bottom. When the CO2 concentration was higher than 500 ppm, the fan speed was increased by 15%, and when it was lower than 450 ppm, the fan speed was reduced by 10%. After spraying, the mixture was allowed to stand for 45 minutes, maintaining a temperature of 20℃ and a humidity of 72%.

[0128] S6: Six cross-shaped aeration ports were made on the surface of each cultivation bag using a 10mm diameter annular punch, with an opening depth of 2.0cm and a spacing of 7cm between adjacent ports; an ultrasonic atomizing device with a 32kHz atomization frequency and a 15μm atomized particle diameter was configured; an intermittent pulse mode of 10 seconds of spraying followed by 20 seconds of rest was adopted, with the atomization volume controlled at 0.8mL / time·m²; the surface moisture content of the mycelium was monitored in real time using an infrared moisture sensor and stabilized at 68%; the ambient temperature was maintained at 22℃, the relative humidity at 85%, and the CO2 concentration at 450ppm.

[0129] S7: Construct a three-layer temperature and humidity gradient in the vertical direction of the cultivation rack, with the top layer temperature at 22℃, the middle layer temperature at 20℃, and the bottom layer temperature at 18℃; set the humidity at the top layer to 88%, the middle layer humidity at 82%, and the bottom layer humidity at 78%; install adjustable ventilation holes on the side walls of the cultivation rack, with the ventilation openings depending on the layer: 40% for the top, 30% for the middle, and 20% for the bottom, using this airflow organization to form a natural convection microenvironment from top to bottom to guide the uniform development of fruiting bodies;

[0130] S8: When the fruiting body cap diameter reaches 3.8cm and the edge changes from inward rolling to flat, it is considered to have met the harvesting standard; start the refrigeration unit to pre-cool the airflow to 6℃ lower than the cultivation environment, adjust the wind speed to 1.2m / s, and blow the airflow downward at a 45° angle onto the cultivation rack; the drying process is carried out in two stages, the first stage lasts for 6 hours, the airflow temperature is maintained at 18℃, and the second stage lasts for 12 hours, the air temperature is adjusted to 16℃; monitor the moisture content of the stipe in real time using a near-infrared moisture detector, and stop the drying process when the moisture content drops to 70%; finally, use a rotating cutting knife to harvest at 1.0cm from the surface of the cultivation bag, and immediately transfer the harvested fruiting bodies to a 6℃ pre-cooling temporary storage room for preservation.

[0131] Table 1 Comparison of parameters in each embodiment ;

[0132] As can be seen from Table 1 above, Example 1 still performs best in many key performance indicators, demonstrating its outstanding advantages in energy saving, environmental stability, and intelligent operation. Specifically, Example 1 consumes approximately 10.8% less energy and 11.3% less water than Example 2, achieving precise resource release and conservation through more efficient blue light control and atomization logic. It exhibits minimal environmental control fluctuations, with temperature fluctuations of ±0.2℃ and humidity fluctuations of ±1.1%, far lower than the other two groups, demonstrating excellent microclimate stability, which is beneficial for stabilizing mycelial growth rhythm and mushroom quality. Its superior intelligent response performance, with a CO2 concentration response delay of only 14 seconds and a color-changing success rate of up to 96.5%, indicates that the airflow organization, enzyme activation, and sensor feedback chain of Example 1 are more efficient. Furthermore, it boasts high management efficiency and low labor intensity, requiring only 1.2 manual intervention operations per day on average, far lower than Example 2, reflecting its superior automation and standardization, making it suitable for large-scale factory production.

[0133] In summary, Example 1 not only surpasses other solutions in product quality, but also boasts significant advantages in resource utilization efficiency, cultivation stability, and intelligent operation and maintenance capabilities. It is recommended as the preferred path for the industrial-scale promotion of efficient three-dimensional cultivation methods for velvet mushrooms.

[0134] Table 2 Comparison of the performance of finished products of *Deer Antler Mushroom* ;

[0135] As shown in Table 2 above, Example 1 outperforms the other two groups in key indicators such as fruiting cycle, unit yield, effective fruiting body rate, commercialization rate, and sensory score, resulting in the best overall product quality. Specifically, Example 1 employs precise blue light wavelength (455nm), reasonable light duration (11h / d), and dynamic temperature drop strategy (0.8℃ / d), which is beneficial for accelerating mycelial growth and metabolic product accumulation. The vertical multi-layered temperature and humidity environment is clearly defined, and combined with dynamic adjustment of the guide plate angle and real-time control of CO2 concentration, it effectively improves the utilization rate of cultivation space and gas exchange efficiency. The enzyme activity control, airflow organization, and atomization frequency design are more scientific, significantly reducing the abnormality rate during fruiting body color change and bud induction stages. The two-stage airflow drying temperature curve is reasonable, with the termination point controlled at a stipe moisture content of 69%, combined with rotary cutting operations to improve post-harvest uniformity and storage quality. In conclusion, Example 1 represents the current optimal cultivation process, and its optimized parameter combination has good application value in actual production.

[0136] This invention encompasses any substitutions, modifications, equivalent methods, and solutions made within the spirit and scope of this invention. To provide the public with a thorough understanding of this invention, specific details are described in detail in the following preferred embodiments; however, those skilled in the art will fully understand the invention even without these details. Furthermore, to avoid unnecessary misunderstanding of the essence of this invention, well-known methods, processes, procedures, components, and circuits are not described in detail.

[0137] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A highly efficient three-dimensional cultivation method for *Pleurotus ostreatus* based on multi-stage temperature and humidity control, characterized in that... Includes the following steps: S1, Substrate pretreatment: The cultivation substrate is crushed and mixed with sawdust, wheat bran, soybean meal and calcium carbonate, and then steam sterilized to obtain the pretreated substrate; S2, Mycelial Culture Stage: The pretreated substrate after inoculation is placed in a three-dimensional cultivation rack culture chamber, and dynamic temperature drop culture is carried out. At the same time, intermittent blue light irradiation is performed, and laccase activity and extracellular polysaccharide content are detected by a metabolite sensor to generate mycelial maturity index. S3, Mycelial Physiological Activity Detection: When the mycelial maturity index reaches the set threshold, the mycelial ATP content is measured. Cultivation bags that meet the requirements enter the next stage, while those that do not meet the requirements are returned for continued cultivation. S4, Dynamic control of environmental parameters: The three-dimensional cultivation rack is divided into different temperature and humidity control zones according to the mycelial maturity index, and an interlayer environmental gradient is constructed by controlling the temperature difference between layers and adjusting the flow plate. S5, Enzyme induction treatment during the color change period: Spray a compound enzyme inducer on the surface of the cultivation bag in the mature area, and at the same time start the longitudinal airflow circulation to maintain the CO2 concentration within the target range; S6, Targeted bud induction treatment: Open oxygen vents on the surface of the cultivation bag after color change, and use intermittent atomization to maintain the moisture on the surface of the mycelium; S7, Gradient Development Regulation: Establish temperature and humidity gradients in the vertical direction of the three-dimensional cultivation rack to promote uniform development of fruiting bodies; S8, Pre-harvest conditioning: When the fruiting bodies reach the harvesting standard, they are dried using low-temperature airflow to regulate the moisture content of the stipe before harvesting.

2. The efficient three-dimensional cultivation method for *Pleurotus ostreatus* based on multi-stage temperature and humidity control according to claim 1, characterized in that, S1 specifically includes: S11, Raw material crushing and processing: The sawdust is crushed to a particle size of 2-3mm using a twin-shaft crusher, and the bran and soybean meal are passed through a 40-mesh sieve respectively; S12, Matrix formulation and mixing: Mix 65% sawdust, 20% wheat bran, 12% soybean meal and 3% calcium carbonate evenly by mass percentage to form a mixed matrix; S13, Pulse Steam Sterilization: The mixed substrate is loaded into the culture chamber, saturated steam is introduced to raise the pressure to 0.15-0.18 MPa, and maintained for 35-45 minutes. After sterilization, the pressure is naturally reduced to normal pressure. S14, Moisture content control: Spray sterile water onto the sterilized substrate to adjust the moisture content to 62-65% to obtain the pretreated substrate.

3. The efficient three-dimensional cultivation method for *Pleurotus ostreatus* based on multi-stage temperature and humidity control according to claim 1, characterized in that, S2 specifically includes: S21, Loading the culture chamber: Spread the pretreated substrate after inoculation evenly in the culture chamber of the three-dimensional cultivation rack, and control the thickness of the substrate layer to 8-10cm. S22, dynamic temperature drop culture: maintain the culture chamber temperature at 24-26℃ for the first 5 days, and then decrease the temperature by 0.7-0.9℃ daily from the 6th day until the temperature drops to 18-19℃, while maintaining the air humidity at 75-78% throughout the process; S23, Intermittent Blue Light Control: Turn on the top-mounted LED light source for blue light irradiation daily, with a wavelength of 450-460nm and a light intensity of 750-850lux. The total irradiation time is 10-12 hours per day, and the light source is turned off for 0.5 hours after every 2 hours of irradiation. S24, Metabolite detection: Laccase activity is detected every 24 hours using a laccase activity sensor at the bottom of the culture chamber, while the extracellular polysaccharide content is detected simultaneously using a microfluidic chip. When the laccase activity is ≥35U / g and the extracellular polysaccharide is ≥18mg / g, a signal indicating that the mycelial maturity has been achieved is generated.

4. The efficient three-dimensional cultivation method for *Pleurotus ostreatus* based on multi-stage temperature and humidity control according to claim 1, characterized in that, S3 specifically includes: S31, Mycelial Sample Collection: After the mycelial maturity standard signal is triggered, take a 0.5-1.0g sample from the middle of the mycelial layer of the cultivation bag, at a sampling depth of 1 / 3 of the substrate thickness. S32, Sample pretreatment: Mix the sample with sterile physiological saline at a mass ratio of 1:4, centrifuge at 8000-10000 rpm for 10-15 minutes at 4-6℃, and take the supernatant. S33, ATP content detection: Add ATP lysis buffer to the supernatant, with a volume ratio of lysis buffer to supernatant of 1:

2. After shaking and mixing, let stand for 5-8 minutes, then inject into a bio-fluorescence detector to measure the fluorescence value. S34, Threshold determination: When the detected ATP content is ≥1.0×10⁻ 8 When the concentration is less than mol / g, it is marked as a qualified cultivation package; the detected value is <1.0×10⁻ 8 Individuals with a concentration of mol / g are marked as not meeting the standard; S35, Re-cultivation treatment: The substandard cultivation package was returned to the S2 stage for 3 more days of cultivation, during which 0.5 hours of blue light irradiation was added daily, and the cultivation temperature was reduced by 0.3℃ compared to the original stage.

5. The efficient three-dimensional cultivation method for *Pleurotus ostreatus* based on multi-stage temperature and humidity control according to claim 1, characterized in that, S4 specifically includes: S41, Cultivation rack zoning: The vertical cultivation rack is divided into three control zones: the upper zone is the active zone, with a height of 1.2-1.5m; the middle zone is the transition zone, with a height of 0.8-1.2m; and the lower zone is the maturity zone, with a height of 0.5-0.8m. S42, temperature and humidity parameter settings: active zone set temperature 20℃, relative humidity 80%, transition zone set temperature 19℃, relative humidity 75%, mature zone set temperature 18℃, relative humidity 70%; S43, Baffle Angle Adjustment: The angle of the interlayer baffle is automatically adjusted according to real-time CO2 concentration data. When the CO2 concentration is >500ppm, the baffle opening angle increases by 5-10°; when the CO2 concentration is ≤450ppm, the baffle opening angle decreases by 5-10°. S44, Environmental Parameter Monitoring: Collect temperature, humidity and CO2 concentration data for each control zone every 30 minutes. Temperature measurement accuracy is ±0.1℃, humidity measurement accuracy is ±1%, and CO2 concentration measurement range is 0-2000ppm.

6. The efficient three-dimensional cultivation method for *Pleurotus ostreatus* based on multi-stage temperature and humidity control according to claim 1, characterized in that, S5 specifically includes: S51, Preparation of inducer: Mix lignin peroxidase and laccase at a ratio of 1:1.5 enzyme activity units, add 0.05% Tween-80 as a surfactant, and prepare a compound enzyme inducer solution with a total enzyme activity of 0.15-0.25 U / mL; S52, Spraying treatment: Use a rotating atomizing nozzle to evenly spray the surface of the mature cultivation bag, with a spraying volume of 15-20 mL / m², a nozzle rotation speed of 3000-3500 rpm, and an atomized particle size of 50-80 μm. S53, Airflow circulation start: Turn on the axial flow fans at the top and bottom of the three-dimensional cultivation rack to form a longitudinal airflow of 0.5-0.8m / s, with the airflow circulating from top to bottom; S54, CO2 concentration control: The concentration is monitored in real time by infrared CO2 sensors installed on each layer of the cultivation rack. When the detected value is >500ppm, the fan speed is increased by 10-15%, and when the detected value is <450ppm, the fan speed is decreased by 5-10%. S55, Environmental balance maintenance: Keep the environment still for 30-45 minutes after induction treatment, during which time the temperature is maintained at 18-20℃ and the relative humidity at 68-72%.

7. The efficient three-dimensional cultivation method for *Pleurotus ostreatus* based on multi-stage temperature and humidity control according to claim 1, characterized in that, S6 specifically includes: S61, Aeration port opening: Use an 8-10mm diameter ring punch to make cross-shaped aeration ports on the surface of the cultivation bag, with an opening depth of 1.5-2.0cm. Make 4-6 aeration ports on each cultivation bag, and keep the distance between adjacent aeration ports 5-8cm. S62, Atomization Preparation: Configure an ultrasonic atomization device, set the atomization frequency to 28-32kHz, and control the atomized particle diameter to 10-15μm; S63, intermittent atomization control: adopts a pulse mode with a working cycle of 8-10 seconds of spraying and 15-20 seconds of intermittent spraying, and the atomization volume is controlled at 0.5-0.8 mL / time·m²; S64, Moisture Monitoring: Real-time monitoring of mycelial surface moisture content using an infrared moisture sensor, maintaining surface moisture content at 65-68%; S65, Environmental Coordination: During atomization, maintain the cultivation environment temperature at 20-22℃, the relative humidity at 80-85%, and the CO2 concentration at 400-450ppm.

8. The efficient three-dimensional cultivation method for *Pleurotus ostreatus* based on multi-stage temperature and humidity control according to claim 1, characterized in that, Specifically, S7 includes: S71, Gradient Parameter Settings: Set three temperature and humidity gradients in the vertical direction of the three-dimensional cultivation rack. The top temperature is set to 22℃, the middle temperature to 20℃, and the bottom temperature to 18℃; the top humidity is set to 88%, the middle humidity to 82%, and the bottom humidity to 78%. S72, Airflow organization and control: Adjustable ventilation holes are set on the side of the cultivation rack, with the upper ventilation holes opening at 30-40%, the middle at 20-30%, and the lower at 10-20%, forming natural convection from top to bottom.

9. The efficient three-dimensional cultivation method for *Pleurotus ostreatus* based on multi-stage temperature and humidity control according to claim 1, characterized in that, S8 specifically includes: S81, Harvesting criteria: When the diameter of the fruiting body cap reaches 3.2-3.8cm and the edge of the cap changes from inward rolling to flat, it is judged to meet the harvesting criteria; S82, Preparation of airflow drying system: Start the refrigeration unit to pre-cool the airflow temperature to 4-6℃ lower than the ambient temperature, adjust the airflow speed to 0.8-1.2m / s, and convey the airflow downward at a 45° angle to the cultivation rack; S83, gradient drying process: drying is carried out in two stages: the first stage lasts for 1-6 hours, maintaining the airflow temperature at 16-18℃; the second stage lasts for 6-12 hours, reducing the temperature to 14-16℃. S84, Moisture content control: The moisture content of the stipe is monitored in real time by a near-infrared moisture meter, and drying is terminated when the detected value drops to 68-70%. S85, Harvesting operation: Use a rotary cutter to harvest along the surface of the cultivation bag at a depth of 0.5-1.0cm. After harvesting, immediately transfer to a pre-cooling room at 4-6℃ for temporary storage.

Images